The goal of this proposed research is to develop a facile and widely applicable platform for the generation of polysaccharide (PS) conjugated anti-bacterial vaccines with particular target of E. coli O157. Bacterial infections constitute one of the major health problems worldwide. Polysaccharides (or O-antigens), forming a thick capsule that surrounds the bacterial pathogen, represent a major determinant of pathogenicity. With the increasing emergence of resistance toward major antibiotics, development of polysaccharide-based vaccines provides an attractive approach for fighting the infectious diseases. The traditional chemical approach has enabled the production of several highly successful conjugated vaccines that are currently in clinical use. However, the traditional approach suffers from multiple fermentations, purification steps, low yields, non-specific chemical conjugation, and therefore, ultimately leading to the high cost of vaccine production. The objective of this proposal is to explore a recently established bacterial protein N-glycosylation system to obtain polysaccharide conjugate vaccines in a facile, efficient, and easily applicable manner. This bacterial glycosylation system comprises a highly promiscuous protein PglB, which catalyze the transfer of polysaccharides from a diphospho-lipid donor to target proteins. The proof-of-concept experiments conducted by us and other groups lend compelling evidence for further exploration of such a system in the conjugate vaccine development. In this Phase I SBIR grant, we plan to demonstrate the applicability of this system by generating polysaccharide conjugate vaccines against E. coli O157: Aim 1: We will validate the PglB-mediated glycosylation of vaccine carrier protein in vitro. Aim 2: We will construct an engineered E. coli host strain for in vivo generation of polysaccharide protein conjugates. Aim 3: We will carry out bioassay to evaluate the conjugate vaccines. We expect the success of this program will allow us to develop a technological platform to produce polysaccharide conjugate vaccines in an efficient, widely applicable manner. PUBLIC HEALTH RELEVANCE: Bacterial infections constitute one of the major health problems worldwide, and are one of the major causes of deaths in the infants, young children and the elderly. With the emerging antibiotic resistance developed by an increasing number of bacterial pathogens, development of anti-bacterial vaccines has been actively pursued in recent decades. Lipopolysaccharide (LPS) is the major component of outer leaflet of many pathogenic bacterial species, such as E. coli O157. LPS plays an essential role as mediator between bacterial cells and the environment. It has been recognized as an important pathogenic factor. Several LPS based vaccines are currently in development or in clinical trials, which shows promising results towards bacterial infection. In this program, we develop a novel system to produce conjugate vaccines in a facile and widely applicable manner compared to the traditional chemical approach.